Isolation and genetic characterization of metallo-β-lactamase and carbapenamase producing strains of Acinetobacter baumannii from patients at Tehran hospitals.

Background and Objective Carbapenems are therapeutic choice against infections caused by gram-negative bacilli including strains of Acinetobacter baumannii. Resistance to these antibiotics is mediated by efflux pumps, porins, PBPs and ß-lactamases. The aim of this study was to determine the possibility of existence of MBLs, OXAs and GES-1 betalactamase genes among clinical isolates of Acinetobacter collected from Tehran hospitals. Material and Methods Two hundred and three Acinetobacter isolates were collected from patient at Tehran hospitals. The isolates were identified using biochemical tests. The susceptibility to different antibiotics was evaluated by disk diffusion method and MICs of imipenem were determined using Micro broth dilution method (CLSI). PCR was performed for detection of bla VIM-2, bla SPM-1, bla IMP-2, bla GES-1, bla OXA-51, bla OXA-23 betalactamase genes. Clonal relatedness was estimated by PFGE with the restriction enzyme SmaI. Results and Conclusion Of 100 isolates of imipenem resistant Acinetobacter spp. collected from Tehran hospitals in 2009 and 2010, 6 isolates produced metallo-beta-lactamases and 94 isolates produced OXA-type carbapenemase. The bla SPM-1, bla GES-1, bla OXA-51, bla OXA-23 genes were detected by PCR among 6, 2, 94 and 84 isolates of A. baumannii, respectively. The MICs of isolates to imipenem were 8–128 µg/mL. PFGE analysis of 29 bla OXA-51 and bla OXA-23-positive A. baumannii isolates gave 6 different patterns. This is the first report of SPM-1 and GES-1 beta-lactamase producing A. baumannii. Production of the OXA-23, OXA-51, GES-1 and SPM-1 enzyme presents an emerging threat of carbapenem resistance among A. baumannii in Iran.


INTRODUCTION
Acinetobacter is an important cause of nosocomial infections and has been associated with a wide variety of illnesses in hospitalized patients, especially patients in the intensive care units (1). The clinical strains of A. baumannii are usually multidrug resistant to aminoglycosides, fluoroquinolones, ureidopenicillins and third generation cephalosporins. In cases of resistance to betalactams caused by extended spectrum β-lactamases and AmpC enzyme, carbapenems are often used as the last resort against infections. However, carbapenem-hydrolyzing-β-lactamases of molecular class B and D have emerged over the last decade (2).
Class B carbapenemases including IMP and VIM termed as metallo beta lactamases (MBLs) have been found so far in A. baumannii and these are encoded by different plasmid types (3). Class D (OXA) have emerged as the major carbapenemases in the world. OXA enzyme (encoded by bla OXA genes) can be subclassified into eight subgroups, of which OXA-23like, OXA-24-like, OXA-51-like and OXA-58-like have been identified in A. baumannii. The bla OXA-51type genes are intrinsically harbored by A. baumannii isolates (4). Recently, new classes of betalactams including SPM-1, GIM-1, SIM, and AIM have also been reported. GES-2, GES-4 and GES-5 class A β-lactamases showed weak activity for hydrolysis of imipenem (5).
The first nosocomial outbreak with carbapenemresistant A. baumannii) CRAB )was reported from the USA in 1991. Since then, CRAB infections and extensive hospital outbreaks have been reported throughout the world (6). However, despite the worldwide occurrence of epidemic carbapenem resistance strains, metallo beta lactamase producing A. baumannii isolates have been found to be disseminated only in specific geographic areas (7). Gram negative bacilli producing acquired IMP, VIM and SPM-1 has been reported more in Asia and South America, respectively (8). Because of the importance of resistance to beta lactam antibiotics among clinical isolates, understanding the underlying genetic mechanisms responsible for the acquisition and spread of unique beta lactamase-mediated antibiotic resistance mechanism could eventually facilitate the development of effective prevention and control measures (9).
Recent studies have shown the existence of different MBLs producing clones of A. baumannii in Iran. The aims of this study were to determine the drug susceptibility patterns of Acinetobacter spp. isolated from patients at different Tehran hospitals and to identify the genes encoding VIM, IMP, SPM and GES carbapenemase among the isolates. Pulsed-Field Gel Electrophoresis (PFGE) was then used to investigate the genetic relationships among the isolates and possibility of intra-and inter-hospital spread of resistant strains.

Bacterial identification and susceptibility testing.
Two hundred and three non-repetitive isolates of Acinetobacter recovered from blood, wound, urine, sputum, and respiratory tract were obtained from 7 hospitals in Tehran between May 2009 to September 2010. The isolates were identified by conventional biochemical methods and confirmed by PCR assay targeting the bla OXA-51 like gene (10).
Strains found resistant to imipenem by disk diffusion test were re-checked in broth micro dilution assay . MIC for imipenem ranging from 0.25 µg/ml through 128 µg/ml was tested. ATCC Pseudomonas aeruginosa 27853 was used as control strain. The MICs ≥ 8 µg/ml was interpreted as resistance to imipenem (13). PCR assay. DNA template from imipenem resistant isolates (MIC ≥ 8 µg/ml) were extracted by boiling for 15 minutes and used as template in PCR assay to amplify bla VIM-2 , bla SPM-1 , bla IMP-2 , bla GES-1 , bla OXA-51 , bla OXA-23 genes by using thermocycler (Eppendorf, Hamburg, Germany). The primers described by Shibata, Weldhagen and Woodford were used in this experiments (14,15,4). The reactions were initiated in solution containing 200 µM concentrations of dNTPs, 10 pM of each primer, 0.8 mM MgCl 2 , 0.5U Taq polymerase (Metabion, Martinsried, Germany) and 50 ng DNA template in a final volume of 25 µL.
plasmid DNA extraction and conjugation experiments. Plasmid DNAs from the bla SPM and bla GES positive strains of A. baumannii were extracted using the plasmid extraction kit (Qiagen, Courtaboeuf, France) and targeted for the bla SPM and bla GES-1 genes using the primers described previously. Transfer of resistance by conjugation was attempted using A. baumannii (donor) and E. coli K12 (recipients). Overnight filter mating experiments were performed at 37ºC, and the transconjugants were selected on MacConkey agar and plates supplemented with imipenem (16 μg/ml).

MBL screening.
Screening for MBLs in selected isolates was performed with combined disk synergy test. In brief, disks containing 750 µg of EDTA plus 10 µg of imipenem were placed on the inoculated plates containing Muller Hinton agar. An increase of ≥ 17 mm in zone diameter in the presence of 750 µg of EDTA compared to imipenem alone indicated the presence of an MBL (9).

PFGE.
The intact chromosomal DNAs from 29 MDR imipenem-resistant Acinetobacter baumannii carrying bla OXA-23 )MIC IMP ≥ 8 µg/ml) belonging to the same antibiotype were extracted for PFGE as describ-ed by Durmaz et al. (17). These strains collected from 5 hospitals where the most collected isolate came from. The PUlseNet universal standard marker strain Salmonella choleraesuis serotype Branderup H9812 was used as a molecular size marker. The gels were stained with ethidium bromide and DNA patterns were photographed with UVP gel Documentation (UVP, UK) (Fig. 1). The DNA banding patterns were analyzed using GelCompare II software (Apllied maths NV, St-Martens-Latem Beligum). PFGE DNA pattern was compared and interpreted according to criteria of Tenover et al. .

Statistical Analysis.
Description and analysis of the data were carried out using SPSS 16.0 software. The nonparametric Mann-Whitney test was used for comparison between the different groups. A p-value ≤ 0.05 was considered significant. RESUlTS
A total of 100 selected isolates were examined for MBL profiles by the combined-disk method. This method showed that 9% of isolates were able to produce MBLs, of which 6 strains were identified as A. baumannii and 3 isolates belonged to the other species of Acinetobacter.
All of the bla SPM positive isolates were collected from different hospitals and their antibiogram indicated that our studied isolates were resistant to all studied antibiotic except polymixin B and colistin. Only two isolates (ABI/43 and ABI/51) carried bla SPM , bla GES , bla OXA-51 and bla OXA-23 simultaneously (   Sequencing. Aligning of the obtained sequences with those of reference strains in Gene Bank confirmed the correct identification of bla GES-1 , bla SPM-1 , bla OXA-51 and bla OXA-23 by PCR.

PFGE analysis.
Totally, 29 isolates with MDR phenotype were analyzed by PFGE and these were collected from hospitals A (n = 13), B (n = 7), C (n = 6), D (n = 2) and E (n = 1). They showed co-resistance to antimicrobial agents including amikacin, aztreonam, piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, cefexime, cefotaxime, cefteriaxone, ciprofloxacin, meropenem, polymyxin B and colistin. Genotyping identified 6 distinct pulsotypes allocated as A to F among 5 hospitals (Fig. 1). The pulsotypes A, B and C were the dominant types found in the hospitals. Conversely, pulsotypes D, E and F each contained single isolate. The pulsotype A consisted of 19 isolates. They were differentiated into closely related pattern comprising of patterns A1-A10 (Fig.  1). Isolates from hospital A were heterogeneous since 7 different banding patterns were found for 13 isolates. More diversity were found among the isolates at hospitals B (n = 7) and C (n = 6) since 6 and 4 pulsotypes were found among the isolates of these hospitals respectively (Fig. 1). Six SPM-1-producing and two GES-1-producing isolates produced closely related patterns.

DISCUSSION
This is the first report on the existence of bla SPM-1 and bla GES-1 among the clinical strains of A. baumannii in Iran. Occurrence of SPM-1 MBL among P. aeruginosa has been described among clinical isolates in Brazil. SPM-1 is distinct from VIM and IMP and represents a new subfamily of mobile MBL which is carried on a plasmid. This is important since it can transform both E. coli and P. aeruginosa to ceftazidime resistance (19). Detection of GES-1 among the Iranian strains of A. baumannii is another important finding of the current study. This ESBL enzyme was initially described from a Klebsiella pneumoniae strain in 1998. GES-1 is weakly related to the other Ambler class A β-lactamases, particularly to the plasmid-located ESBLs so far identified in Enterobacteriacae. However, GES-1 may be classified rather as a ceftazidime-hydrolyzing enzyme (20). Our survey showed that these two genes can not be transferred to E. coli K12 strain by conjugation.
While IMP-2 is reported in A. baumannii from Italy and Japan, VIM enzymes have been identified very rarely in A. baumannii, being represented only by VIM-2 reported in South Korea. Despite carbapenemase activity of resistant strains, these MBL genes have not been detected in A. baumannii from Iran (21). Among MBL genes, only VIM-type had been detected in P. aeruginosa in Iran (16).
The MBLs can hydrolyze all beta-lactams except aztreonam. However, all MBL producing isolates in our study were resistant to aztreonam too. Other mechanisms such as ESBL production, efflux pumps and hyper-production of cephalosporinase possibly are involved in resistance to aztreonam in our collection of isolates.
This study showed low susceptibility rates to most of the clinically available antimicrobial agents for the treatment of infections caused by Acinetobacter spp. except for polymyxin B and colistin. This finding suggests both of them as therapeutic options for the treatment of infections caused by these isolates. Among imipenem resistant isolates, 94% harboured at least one acquired class D carbapenemaseencoding gene. Therefore, the high prevalence of class D carbapenemase-encoding genes among the clinical isolates from Iran appears responsible for low susceptibility rates for imipenem [P < 0.05; sig = 0.002]. According to the SENTRY report distribution of OXA-type genes among Acinetobacter spp. isolates in Asia-Pacific nations was comprised mainly of bla OXA-23 , while bla OXA-24/40 and bla OXA-58 were less common. In addition, clonal dissemination was found among different medical centers in different countries in the Asia-Pacific region (22). In our study, bla OXA-23 was the most common genes encoding carbapenamase at Tehran hospitals.
PFGE analysis revealed genetic diversity among the MDR A. baumannii isolates. These results may support the assumption that MDR strains are emerged more by horizontal transfer of MBL genes among different phenotypic clones of Acinetobacter. Six SPM-1-producing and two GES-1-producing isolates showed closely related patterns. Such a correspondence of phenotypic and genotypic characteristic can be attributed to a common clonal origin. Some isolates that gave identical typing results had been isolated from patients of different hospitals at different times. Therefore, these types have simultaneous presence and genetically are stable in community. Moreover, a few of these isolates showed different antimicrobial susceptibility patterns. This caused by transfer of these antibiotic resistance genes to the similar clonal strains through transformation or conjugation. The possibility of inter-hospital transmission of potentially pathogenic bacteria including P. aeruginosa has already been debated in Iran (23).
In conclusion, the rate of resistance to betalactams was high in our study. Since the rate of isolates carrying bla SPM-1 and bla GES-1 was very low, it seems that other mechanisms such as decreased permeability, over expression of efflux pump, production of carbapenemases probably are involved in resistance to betalactams. Understanding the underlying genetic mechanisms responsible for the acquisition and spread of this unique beta lactamase mediated antibiotic resistance mechanism could eventually facilitate the development of effective prevention and control strategies and thereby allowing more effective drug usage and treatment of disease, and reducing resistance development (9).

ACKNOWlEDGMENTS
This study was supported by grants from Pasteur Institute of Iran. The authors thank Fahimeh Shooraj, and Hanieh Noveiri for their technical assistance and also the members of clinical microbiology laboratories of Shariati , Imam Khomeini, Mostafa Khomeini, Tehran Heart Center, Taleghani hospitals for their cooperation. Also we are very grateful to Professor Nordmann and Cristina Lagatolla for providing us standard strains for PCR assay.